ISOELECTRIC-FOCUSING OF PROTEINS AND PEPTIDES IN GEL SLABS AND IN CAPILLARIES

The present review offers a survey of modern isoelectric focusing (IEF ) techniques, including the conventional one in soluble amphoteric buf fers (CA-IEF) and immobilized pH gradients (IPG). In the case of CA-IE F, recent advances on its use as a first dimension for two-dimensional electrophoresis are covered. The properties of isoelectric buffers, i n terms of buffering power and conductivity, are reviewed in view of t heir importance as background electrolytes in capillary zone electroph oresis (CZE). In the case of IPGs, examples are given of the high corr elation between theoretically predicted and experimentally measured pi values. New aspects of IPGs, such as focusing in extremely alkaline p H intervals, and its interfacing with mass spectrometry, are described . In the case of capillary IEF, new aspects of the technique are descr ibed, in particular: (a) how to modulate the slope of the pH gradient for increasing resolution; and (b) how to keep proteins in solution at , and in the proximity of, the pi value. The review ends with an excur sus on the use of isoelectric buffers in capillary zone electrophoreti c separations. Such buffers offer unique advantages: they permit very high voltage gradients (up to 1000 V/cm) and, thus, minimize analysis times (down to a few minutes in 30-35 cm long capillaries). This resul ts in a marked increment in resolution due to minimal diffusion-driven peak spreading. Such buffers are finding unique applications for gene rating peptide maps of tryptic digests of proteins and also in the ana lysis of intact proteins. (C) 1998 Elsevier Science B.V. All rights re served.